The steroidogenic response to ACTH can be separated into acute and subacute aspects. The acute response (sec-min) occurs at the level of the mitochondrion and involves the translocation of cholesterol to the inner mitochondrial membrane followed by the conversion of cholesterol to pregnenolone (rate-limiting). The subacute response (hrs) occurs at the level of the genome and leads to the synthesis of steroidogenic enzymes and cofactors. Both the acute and subacute responses to ACTH are mediated, impart, by cAMP, and both are dependent on the de novo synthesis of protein. A steroidogenic regulatory protein has been proposed but none has been identified as yet. The role of Ca 2+ in ACTH action is complex; Ca 2+- regulated protein kinases and/or phosphatases may be involved. Pregnenolone, the product of the rate-limiting step in steroidogenesis, is poorly soluble in aqueous media and must be translocated between intracellular compartments. Progesterone, which is derived from pregnenolone and closely related, not only serves as a key steroid intermediate, but is also a ligand for a member of the steroid receptor super-family. To examine the complex process of ACTH action, specific intra-adrenal steroid-binding proteins are being investigated. Two such proteins have been detected and characterized by the SACB laboratory: 1) a cytoplasmic pregnenolone-binding protein (P5BP), and 2) a nuclear progesterone-binding protein (p4BP). The P5BP has a Mr of 34 kDa and exists as charge isoforms with PI's of 6.4, 5.5, 5.4 and 5.2. The PI 5.4 isoform may represent the active binding molecule. The binding of pregnenolone to the heat-labile P5BP is regulated by a phosphorylation/dephosphorylation system, and requires a heat-stable cofactor. The P5BP kinase has been partially purified and has been found to be absolutely dependent on Ca2+. P5BP is present only in cells regulated by ACTH and not in steroidogenic cells regulated by other peptide hormones. The content of P5BP in fasciculata cells is increased by ACTH. These findings suggest a functional relationship between P5BP and ACTH. The P5BP has been purified and internal peptide sequences determined following partial tryptic digestion. A full-length P5BP cDNA is currently being cloned. The P4BP has been purified 2000-fold by steroid-affinity chromatography, and photoaffinity labeling has revealed a Mr of 50 kDa. Biochemical and immunological evidence indicate that P4BP is distinct from the classical progesterone receptor which has a Mr of 75 to 115 kDa depending on the species examined. The P4BP has been found to bind in vitro to DNA suggesting a transcriptional regulatory role. P4BP synthesis is stimulated in vivo by ACTH further suggesting a mediating role in ACTH action.